Transferable Streptomyces DNA amplification and coamplification of foreign DNA sequences.

The 8.8-kb amplifiable unit of DNA of Streptomyces achromogenes subsp. rubradiris, AUD-Sar 1, which carries 0.8-kb terminal direct repeats and a spectinomycin resistance determinant, can mediate high-level amplification of an AUD-Sar 1-derived 8.0-kb DNA sequence not only in S. achromogenes but also in the heterologous host Streptomyces lividans. This was seen upon introduction of AUD-Sar 1 into chloramphenicol-sensitive strains of S. lividans via the temperature-sensitive (39 degrees C) plasmid pMT660, which contains the thiostrepton resistance gene tsr. Following the cultivation of transformants at 39 degrees C on media containing spectinomycin, a number of strains which were unable to grow on thiostrepton and which carried the amplified 8.0-kb DNA sequence as arrays of 200 to 300 copies of tandem 8.0-kb repeats were found. Chloramphenicol-resistant strains of S. lividans did not yield amplified sequences under similar conditions. Studies with plasmids carrying inserted antibiotic resistance genes at two sites of AUD-Sar 1 yielded coamplified sequences which contain the inserted DNA. Transformation with a plasmid carrying a 1.0-kb deletion in AUD-Sar 1 followed by growth under similar conditions yielded a 7.0-kb repeated DNA sequence. Southern analysis revealed the absence of vector sequences located on the right side of AUD-Sar 1 in the input plasmids in all examined DNA samples of amplified strains. In contrast, a majority of the samples revealed the presence at unit copy level of AUD-Sar 1 left-adjacent sequences which are part of the input plasmids and in several samples the presence of certain vector sequences located near them. The results suggest input plasmid integration into the S. lividans chromosome prior to the generation of the amplified sequences and the deletion of AUD-Sar 1 adjacent sequences.

DNA amplification in Streptomyces achromogenes subsp. rubradiris is accompanied by a deletion, and the amplified sequences are conditionally stable and can be eliminated by two pathways.

Southern blot analysis of BglII-digested DNA isolated from wild-type Streptomyces achromogenes, which harbors the 8.8-kilobase amplifiable unit of DNA, AUD-Sar 1, and of similarly digested DNA from 12 strains carrying an array of 200 to 300 tandem copies of a specific AUD-Sar 1-derived 8.0-kilobase DNA sequence, ADS-Sar 1, revealed the absence of the 12.4-kilobase BglII AUD-Sar 1-chromosome right junction band in the latter strains, whereas the corresponding 26.0-kilobase left junction band remained unaltered. Further Southern analyses indicated in all of the seven amplified strains tested the occurrence of a deletion of at least 10 kilobases of the DNA adjacent to the right side of the AUD. The deletion has one endpoint in the vicinity of the ADS array. Corroborating and expanding upon previously reported results, we found that the amplified DNA of strain C010 was stably maintained for at least 20 transfers when the transfers involved mycelia propagated in spectinomycin-free liquid medium. In contrast, when strain C010 was subjected separately to one cycle of protoplast formation and regeneration or to three cycles of spore germination, aerial mycelium formation, and sporulation on spectinomycin-free media, only approximately 20% of the protoplast regenerants and spores retained the reiterated DNA sequences and the ability subsequently to form colonies on media containing high levels of spectinomycin. Approximately 80% of these units completely deleted the reiterated DNA and left adjacent sequences and exhibited sensitivity to 25 micrograms of spectinomycin per ml. One among 24 protoplast-derived deletants apparently retained the left portion of the AUD-ADS left direct repeat plus left adjacent sequences.

Spectinomycin resistance and associated DNA amplification in Streptomyces achromogenes subsp. rubradiris.

Streptomyces achromogenes subsp. rubradiris plated at low density on 1,000 micrograms of spectinomycin per ml initially produces slow-growing, bald colonies from which arise, in a spatially and temporally random fashion, foci of rapidly growing aerial mycelium-forming cells whose DNA contains an approximately 200- to 300-fold amplification of an 8-kilobase (kb) sequence. This sequence was cloned in Escherichia coli on pBR322 and physically characterized. It was separately cloned also in Streptomyces lividans as a BglII fragment and shown to impart high-level resistance to spectinomycin in an orientation-independent manner when present in either the high-copy-number vector pIJ702 or the unit-copy-number vector pIJ943. A spectinomycin resistance determinant was shown to reside on a 1.7-kb SphI-BglII subfragment. Analysis of Southern blots of restriction enzyme digests of wild-type S. achromogenes DNA probed with the labeled 8-kb DNA sequence resulted in the identification and subsequent cloning in S. lividans of a 10.4-kb BamHI fragment which probably includes the complete 8.8-kb amplifiable unit of DNA. This unit is present in wild-type S. achromogenes and in the initially slow-growing, bald colonies arising on 1,000 micrograms of spectinomycin per ml as a single copy. It carries two 0.8-kb direct repeats at its termini as well as the spectinomycin resistance determinant close to one of these termini. About 5% of protoplast regenerants from wild-type S. achromogenes and 77% of protoplast regenerants from the rapidly growing strains lost both the ability to grow on spectinomycin at 10 micrograms/ml and the sequences that hybridize with the 8-kb probe DNA. The 1.7-kb Bg/II-SphI resistance fragment, when introduced via the vector pIJ702 into an S. achromogenes strain sensitive to 10 microgram of spectinomycin per ml, permitted its vigorous growth on 1,000 micrograms of the antibiotic per ml.

Lack of influence of the carbamoyl group on the stereochemistry of the acid-catalyzed opening of the aziridine ring of the mitomycins and of congeners.

The acid-catalyzed opening of the aziridine ring of mitomycins A and C is known to occur predominantly with cis stereochemistry. We have observed that the presence or absence of a carbamoyl group at C-10 of mitomycin C and in certain of its analogues does not have a significant influence on the stereochemistry of the opening of this ring. The trans product obtained from mitomycin C was shown to be stable when treated with acid under the conditions of its formation. Mitomycin B was also shown to yield predominantly the cis product when it was subjected to acid-catalyzed opening of its aziridine ring. The 1H NMR spectra of acetate derivatives prepared from mitomycin B show two sets of signals that are due to two populations of rotamers. The analysis of these spectra has substantiated several previous spectral assignments. This paper also presents some thoughts on acid-catalyzed bifunctional DNA alkylation by mitomycins and 10-decarbamoyloxy-9-dehydromitomycins.

Studies on the biosynthesis of the antibiotic streptozotocin (streptozocin) by Streptomyces achromogenes var. streptozoticus. Feeding experiments with carbon-14 and tritium labelled precursors.

Feeding experiments and chemical degradations have shown that D-[1(-14)C,2(-3)H]-and-[1(-14)C,6(-3)H] glucosamine, L-[ureido-14C] citrulline, L-[guanidino-14C] arginine and L-[14CH3] methionine specifically label the glucosamine moiety, the urea carbonyl and the N-methyl group of the antibiotic streptozotocin, respectively. Feeding these precursors in amounts of 5 approximately 10 mumoles per 100 ml of culture medium under conditions where the fermentation yielded approximately 20 mumoles of streptozotocin in 24 hours gave incorporation rates which approached 40%. Upon feeding 100 mumoles of either D-[1(-14)C] glucosamine or L-[ureido-14C] citrulline they were incorporated into newly synthesized streptozotocin essentially without dilution by endogeneous precursors. D-[1(-14)C, 6(-3)H] Glucosamine was incorporated without change in T/C ratio while 20% of the tritium was lost from D-[1(-14)C,2(-3)H] glucosamine, suggesting the possibility that D-glucosamine can partially equilibrate with D-fructose prior to its incorporation.

Nitroalkane oxidation by streptomycetes.

Crude cell-free extracts of nine strains of Streptomyces tested for nitroalkane-oxidizing activity showed production of nitrous acid from 2-nitropropane, 1-nitropropane, nitroethane, nitromethane, and 3-nitropropionic acid. These substrates were utilized in most strains but to a decreasing extent in the order given, and different strains varied in their relative efficiency of oxidation. p-Nitrobenzoic acid, p-aminobenzoic acid, enteromycin, and omega-nitro-l-arginine were not attacked. d-Amino acid oxidase, glucose oxidase, glutathione S-transferase, and xanthine oxidase, enzymes potentially responsible for the observed oxidations in crude cellfree extracts, were present at concentrations too low to play any significant role. A nitroalkane-oxidizing enzyme from streptozotocin-producing Streptomyces achromogenes subsp. streptozoticus was partially purified and characterized. It catalyzes the oxidative denitrification of 2-nitropropane as follows: 2CH(3)CH(NO(2))CH(3) + O(2) --> 2CH(3)COCH(3) + 2HNO(2). At the optimum pH of 7.5 of the enzyme, 2-nitropropane was as good a substrate as its sodium salt; t-nitrobutane was not a substrate. Whereas Tiron, oxine, and nitroxyl radical acted as potent inhibitors of this enzyme, superoxide dismutase was essentially without effect. Sodium peroxide abolished a lag phase in the progress curve of the enzyme and afforded stimulation, whereas sodium superoxide did not affect the reaction. Reducing agents, such as glutathione, reduced nicotinamide adenine dinucleotide, and nicotinamide adenine dinucleotide phosphate, reduced form, as well as thiol compounds, were strongly inhibitory, but cyanide had no effect. The S. achromogenes enzyme at the present stage of purification is similar in many respects to the enzyme 2-nitropropane dioxygenase from Hansenula mrakii. The possible involvement of the nitroalkane-oxidizing enzyme in the biosynthesis of antibiotics that contain a nitrogen-nitrogen bond is discussed.

Isolation and characterization of tryptophan transaminase and indolepyruvate C-methyltransferase. Enzymes involved in indolmycin biosynthesis in Streptomyces griseus.

Two enzymes, tryptophan transaminase and indolepyruvate C-methyltransferase, which are active in the initial steps of the biosynthetic pathway of the antibiotic indolmycin, have been detected and partially purified from cell-free extracts of Streptomyces griseus. The transaminase has been purified 3-fold by ammonium sulfate fractionation. At this stage of purification, it catalyzes the alpha-ketoglutarate and pyridoxal phosphate-dependent transamination of L-tryptophan, 3-methyltryptophan, L-pphenylalanine, and L-tyrosine. The C-methyltransferase catalyzes the transfer of a methyl group from S-adenosylmethionine to position 3 of the aliphatic side chain of indolepyruvate. No cofactors are required. The C-methyltransferase has been purified 110-fold by ammonium sulfate fractionation, Sephadex G-150 gel filtration, DEAE-Sephadex column chromotography, and Bio-Gel A-5m gel filtration. The enzyme has a broad pH optimum of 7.5 to 8.5. A molecular weight of 55,000 +/- 5,000 has been determined by Sephadex G-200 gel filtration with reference proteins and a molecular weight of 58,000 +/- 8,000 has been determined by sucrose density gradient centrifugation. The enzyme is relatively stable at temperatures of 0-5 degrees but is destroyed by freezing or by heating. The C-methyltransferase is inhibited strongly by the thiol reagents p-chloromercuribenzoate and N-ethylmaleimide. The Zn2+ and Fe2+ chelators 1,10-phenanthroline and 2,2'-bipyridine also inhibit the enzyme activity but EDTA does not. Michaelis-Menten constants have been determined for the 110-fold purified enzyme as 1.2 X 10(-5) M for S-adenosylmethionine and 4.8 X 10(-6) M for indolepyruvate. The enzyme activity in the crude extract is inhibited competitively by indolmycin (Ki equals 2.3 mM) and L-tryptophan (Ki equals 0.17 mM), but these effects are not observed after the enzyme has been passed through the Sephades G-150 column during purification. The crude extract is capable of methylating phenylpyruvate and p-hydroxyphenylpyruvate but this capability is lost upon purification of the indolepyruvate C-methyltransferase activity. No methylation of L-tryptophan occurs under the conditions used.